Las 500 image system

Protein concentration in the lysates was determined using the Bradford assay with bovine serum albumin as a reference standard (Pierce™ Coomassie Bradford Protein Assay Kit, Thermo Fisher Scientific). Samples containing equal protein amounts in sample buffer were loaded on 4–15% SDS-PAGE precast gels (Bio-Rad, Hercules, CA, USA) and separated by electrophoresis. After proteins were transferred onto polyvinylidene fluoride membrane, the membranes were blocked with blocking buffer (Tris-buffered saline with 5% non-fat dry milk and 0.01% Tween-20) for 1 h at RT. Then, the membranes were incubated with each primary antibody solution (1:1000 in blocking buffer) at 4 °C overnight. The membranes were washed with Tris-buffered saline with 0.01% Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000 in blocking buffer). Protein signals were visualized using an enhanced chemiluminescence detection reagent. Protein band images were obtained using an LAS 500 image system (GE Healthcare, Chicago, IL, USA). Blot signals were quantitated by densitometry analysis using ImageJ software (NIH, Bethesda, MD, USA). Protein levels were normalized to those of the corresponding loading controls.

Hippocampus and SH-SY5Y cells were lysed by tissue protein extraction reagent (T-PER) (Thermo Scientific, Waltham, MA, USA), or RIPA buffer (Biosesang, Gyeonggi-do, South Korea). After centrifugation (13000× g, 4 °C, 15 min), total protein concentration in the supernatant was measured with a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA, USA). Protein was separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was incubated with 5% non-fat milk (Bio-Rad Laboratories, California, USA) in Tris-buffered saline with Tween-20 (TBST) at 22 °C for 2 h. Primary antibodies were diluted according to the manufacturers’ specifications (Table 1) and incubated at 4 °C overnight. The membrane was washed with TBST and then incubated with secondary antibodies (goat anti-rabbit IgG-HRP) (Thermo Scientific, Waltham, MA, USA) for 2 h. Bands were detected using a clarity western chemiluminescent (ECL) substrate kit (Bio-Rad Laboratories, Hercules, California, USA), and band images were obtained using a LAS 500 image system (GE Healthcare, UK). The bands were quantified with Quantity One software (Bio-Rad Laboratories, Hercules, California, USA).